Dual peptide presentation from bioengineered carriers to potentiate stromal cell function and tissue repair

项目总结 在美国每年发生的600多万例骨折中，5%-20%会导致骨不连或延迟 友联市。基于细胞的疗法代表了传统骨移植或植入物的一种令人兴奋的替代方案，但细胞 移植需要一种可裁剪的底物，为移植的细胞提供必要的线索。间充质 干细胞/基质细胞(MSCs)是一种有吸引力的细胞来源，用于组织工程，因为其强大的 分泌促血管生成和抗炎营养因子。在适当的刺激下，MSCs可以 通过分化为成骨细胞直接促进骨形成，但以成骨方式诱导 MSCs存在促血管生成因子分泌减少的问题。我们演示了一个 将促血管生成多肽Gly-His-Lys(GHK)包埋在藻酸盐水凝胶中，使MSCs的生长速度提高4倍 它们的促血管生成潜力增加。我们之前加入了多肽配体，如Arg-Gly-Asp (RGD)促进细胞与离子交联型海藻酸盐和光交联型海藻酸凝胶(PAHs)的黏附 具有更多受控的降解曲线。RGD促进MSCs的成骨分化，但可能会损害 内源性促血管生成信号的分泌。因此，迫切需要一种能够 同时增强移植的MSCs的促血管生成和成骨能力，以最大限度地提高其 在基于细胞的治疗中的疗效。我们的中心假设是骨髓间充质干细胞可以同时被刺激经历 成骨分化，同时分泌有效的促血管生成信号，转化为强化治疗 通过增加局部血管形成和骨形成来发挥潜力。目的1.确定双肽的作用 包裹在多环芳烃中的MSC成骨分化和促血管生成潜能的信号。我们 将合成不同密度的RGD和GHK的多环芳烃。凝胶的生物物理性质的变化， 此外，还将确定包裹的人MSCs的成骨和促血管生成反应。目标2. 确定多肽递呈多环芳烃的必要生物物理性质以指导MSC成骨 和促血管生成潜能。我们将研究每个肽在成骨分化和成骨中的作用。 促血管生成潜能，同时测量细胞黏附和基质整体硬度对MSC的贡献 回应。目的3.证实骨髓间充质干细胞在双肽修饰中的治疗潜力 藻酸盐可促进啮齿动物临界大小的颅骨缺损的血管形成和骨修复。我们 将表征骨髓间充质干细胞植入多肽递呈多环芳烃促进骨修复的能力 原位缺陷。植入细胞的作用、骨形成的数量和质量将通过 非侵入性成像方式和组织学分析。这项拟议的研究具有创新性，因为它 利用两种不同的多肽的活性和可生物降解的水凝胶来增强修复潜力 骨髓间充质干细胞。这项研究将为调控骨形成提供一种新的方法，并且策略已经 提高以材料为基础的组织修复疗法疗效的潜力。

PROJECT SUMMARY Of the greater than 6 million fractures occurring yearly in the US, 5-20% will result in nonunion or delayed union. Cell based therapies represent an exciting alternative to traditional bone grafting or implants, but cell transplantation requires a tailorable substrate to provide necessary cues to implanted cells. Mesenchymal stem/stromal cells (MSCs) are an attractive cell source for use in tissue engineering because of their robust secretion of proangiogenic and anti-inflammatory trophic factors. Upon appropriate stimulation, MSCs can directly contribute to bone formation by differentiating to bone-forming osteoblasts, yet osteogenically induced MSCs suffer from reduced secretion of proangiogenic factors. We demonstrated that the presentation of a proangiogenic peptide, Gly-His-Lys (GHK), to MSCs entrapped in alginate hydrogels resulted in up to a 4-fold increase in their proangiogenic potential. We previously incorporated peptide ligands such as Arg-Gly-Asp (RGD) to facilitate cell adhesion to ionically-crosslinked alginate and photocrosslinkable alginate gels (PAHs) with more controlled degradation profiles. RGD stimulates osteogenic differentiation of MSCs but may impair secretion of endogenous proangiogenic cues. Thus, there is a pressing need for biomaterials that can simultaneously enhance the proangiogenic and osteogenic potential of transplanted MSCs to maximize their efficacy in cell based therapies. Our central hypothesis is MSCs can be simultaneously stimulated to undergo osteogenic differentiation while secreting potent proangiogenic cues, translating to enhanced therapeutic potential by increasing local vascularization and bone formation. Aim 1. Determine the role of dual peptide signaling on MSC osteogenic differentiation and proangiogenic potential when entrapped in PAHs. We will synthesize PAHs with varying densities of RGD and GHK. Changes in the biophysical properties of the gel, as well as the osteogenic and proangiogenic response of entrapped human MSCs will be determined. Aim 2. Define the necessary biophysical properties of peptide-presenting PAHs to instruct MSC osteogenic and proangiogenic potential. We will examine the role of each peptide on osteogenic differentiation and proangiogenic potential, while measuring the contributions of cell adhesion and substrate bulk stiffness to MSC response. Aim 3. Demonstrate the therapeutic potential of MSCs deployed in dual peptide-modified alginate to promote vascularization and bone repair in rodent critical-sized calvarial bone defects. We will characterize the capacity of MSCs implanted in peptide-presenting PAHs to promote bone repair in an orthotopic defect. The role of implanted cells, quantity, and quality of bone formation will be assessed using noninvasive imaging modalities and histological analysis. The proposed research is innovative because it exploits the activity of two distinct peptides with a biodegradable hydrogel to potentiate the reparative potential of MSCs. This research will provide a novel approach for regulating bone formation, and the strategies have potential in enhancing the efficacy of materials-based therapies for tissue repair.